Identification and functional characterization of a Na +-independent large neutral amino acid transporter (LAT2) on ARPE-19 cells

Abstract

The objective of this study was to investigate the presence of a large neutral amino acid transporter on the ARPE-19 cell line. ARPE-19 cells were grown on 24-well plates for uptake studies. Uptake characteristics of [ 3 H ] l-phenylalanine ( l-Phe) were determined at various concentrations and pH at 37 °C. Inhibition studies were conducted in presence of l- and d-amino acids, metabolic inhibitors, like ouabain, sodium azide, and in presence of sodium-free medium, to delineate the mechanism of uptake. RT–PCR was carried out on total RNA isolated from the ARPE-19 cells. Presence of Na +-free buffer did reduce the uptake rate. Hence, all experiments were carried out in Na +-free medium to delineate the sodium-independent uptake mechanism. Uptake of l-Phe on ARPE cells was found to be saturable with a K m=89.35±14 μM, V max=58.9±2.5 pmol min −1 mg protein −1, and K d=0.108±0.04 μl min −1 mg protein −1. Dose-dependent inhibition was observed with increasing concentrations of unlabeled l-Phe. Uptake also was found to be energy independent. Significant inhibition of [ 3 H ] l-Phe was observed with large neutral aromatic and aliphatic amino acids as well as small neutral amino acids. System L-specific inhibitor BCH produced partial inhibition of uptake. Neither acidic nor basic amino acids altered the uptake rate. Results obtained were predominantly characteristic of LAT2, particularly with respect to substrate selectivity and pH dependence. Bands for LAT2 were detected by RT–PCR in the ARPE cell line. This study provides biochemical evidence of the presence of a Na +-independent, facilitative transport system, LAT2, on the ARPE-19 cells.